Undergoing lignin-coated seeds to cold plasma to enhance the growth of wheat seedlings and obtain future outcome under stressed ecosystems

Climate changes threat global food security and food production. Soil salinization is one of the major issues of changing climate, causing adverse impacts on agricultural crops. Germination and seedlings establishment are damaged under these conditions, so seeds must be safeguard before planting. Here, we use recycled organic tree waste combined with cold (low-pressure) plasma treatment as grain coating to improve the ability of wheat seed cultivars (Misr-1 and Gemmeza-11) to survive, germinate and produce healthy seedlings. The seeds were coated with biofilms of lignin and hash carbon to form a protective extracellular polymeric matrix and then exposed them to low-pressure plasma for different periods of time. The effectiveness of the coating and plasma was evaluated by characterizing the physical and surface properties of coated seeds using X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), nuclear magnetic resonance (NMR) spectroscopy, and wettability testing. We also evaluated biological and physiological properties of coated seeds and plants they produced by studying germination and seedling vigor, as well as by characterizing fitness parameters of the plants derived from the seeds. The analysis revealed the optimal plasma exposure time to enhance germination and seedling growth. Taken together, our study suggests that combining the use of recycled organic tree waste and cold plasma may represent a viable strategy for improving crop seedlings performance, hence encouraging plants cultivation in stressed ecosystems.

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Introduction
Agricultural wastes contribute to an increase in greenhouse gases, especially CO2, which is responsible for global warming.It has been documented that using the raw materials of plant residues as soil amendments caused increase in CO2 and N2O fluxes (Rizhiya et al., 2011;Toma and Hatano, 2007).There is a positive correlation between the amount of plant raw materials and gas emissions, sharing in climate changes issues (Iles, 2015).In order to reduce gaseous emissions, agricultural wastes should be converted into useful byproducts.In this respect, using these by-products instead of raw wastes showed reductions in gases emissions amounted to 70% (Case et al., 2012;Felber et al., 2014;Kammann et al., 2012).Lignocellulose is the most abundant renewable raw material, but only about 4.4% of it is exploited annually (Abo et al., 2019).Lignin, a significant component in lignocellulosic materials, is an amorphous heteropolymer contains phenylpropane units linked by diversified bonds (Chandra and Madakka 2019;Geng et al. 2019).Despite there are several researches pointed out the beneficial uses and recycling of lignocellulosic materials, knowledge about the importance of lignin as a seed coating is unavailable On the other hand, saline soil environment is a serious challenge for agriculture in arid regions with limited access to freshwater.These regions commonly encounter harmful ecological issues such as drought, extreme heat, nutrient shortages, and soil salinization (El-Bially et al., 2022a, El-Mageed et al., 2022, El-Metwally et al., 2021, Saudy et al., 2019, Saudy et al., 2021b).Such stresses correlate with poor crop growth, development and yields (El-Bially et al., 2022b, El-Metwally and Saudy, 2021b, El-Metwally et al., 2021, Saudy et al., 2021a) and are expected to have a greater effect with global warming, risking food shortages (El-Metwally and Saudy, 2021a, Mubarak et al., 2021, Salem et al., 2021).Three main crop species, wheat (Triticum aestivum), rice (Oryza sativa), and maize (Zea mays) are widely grown around the world and feed more than 50% of the global population.However, present crop breeding technologies and artificial selection are insufficient to tackle the booming worldwide population and deteriorating arable farmland.To solve this issue, new strategies, such as novel smart agriculture approaches, are required to enhance the ability of agricultural plants to adapt to harsh environments.
NTP treatment is a novel class of ecologically benign methods for the secure and efficient surface decontamination and protection of crop seeds.Numerous studies have already investigated the impact of NTP on the chemistry and morphology of the seed, as well as on DNA, plant resilience to drought, and metal toxicity [Starič et al., 2020], suggesting that NTP treatment improves seed properties.Thus, plasma-treated seeds are viewed as a promising option for farming in challenging environments and soils with abnormal salinity (Alves-Junior et al., 2020, Dauwe et al., 2021, Holc et al., 2022, Pérez-Pizá et al., 2019, Shapira et al., 2019, Starič et al., 2020).However, as NTP generates a wide range of positively and negatively charged particles, and reactive oxygen and nitrogen species that may have side effects to seeds on the molecular (DNA) level, care needs to be taken to optimize plasma exposure times.On the other hand, shorter exposure times may not be sufficient to produce beneficial effects.Therefore, one of the remaining challenges in the field is the need to optimize the plasma treatment time to minimize potential unwanted effects of the NTP and decrease the costs, while maintaining the beneficial effects of seed treatment.
To address this challenge and enhance seed functionality, in the present study we made use of recycled tree waste (modified lignin and lignocellulosic material) (Söylemez et al., 2021) to be exploited in coating the wheat seed surface with a protective layer prior to NTP treatment.Lignin is a complex phenolic polymer, and the NTP treatment is expected to enhance its rigidity, the hydrophobicity of the plant cell wall, and the transport of minerals across vascular bundles in plants, thus protecting the seed from salinity.To test these hypothesizes; we focused on the analysis of two wheat varieties that are frequently used in arid regions with limited access to fresh water, i.e.Egyptian wheat seeds Misr-1 and Gemme-za-11.We examined how treatment with different doses of NTP affects the lignocellulosic biomaterial coating thicknesses around Misr-1 and Gemmeza-11 seeds, and how the coating thickness affects the seeds.Furthermore, after treatment, seed germination and seedling growth and physiology were measured.

Plant material
In every trial, we used seeds from two different wheat cultivars (Cultivar 1: Misr-1 and Cultivar 2: Gemmeza-11).As a control (D(Control)), The control seeds were not treated with chemical lignocellulosic biomass materials and plasma.The lignocellulosic biomass material (prepared as described in 2.2) treatment was done.
Viscous lignin and hash carbon are mixed, and then wheat seeds are added.This material is gently mixed to create a homogeneous surface around the seeds.To protect the seeds from the wet environment, the lignin material containing hash carbon needs to be viscous and non-liquid.The seeds are left in the sun for a day to allow the surface-treatment material to dry.We start preparing the seeds for plasma exposure at different times.
After the seeds were coated with biofilms of lignin and hash carbon to form a protective extracellular polymeric matrix, they were exposed to low-pressure plasma.The low-pressure plasma treatments were done over one of four time periods: one minute (D(00)), two minutes (D( 22)), three minutes (D(33)), or four minutes (D(44)), as described in 2.3.

Chemical lignocellulosic biomass materials
Lignocellulosic biomass in crushed form (roughly ~4 mm in size) was purchased from a mill and then sieved to produce a powder with particle sizes of 250-500 μm.Flavone ligninocellulose (FLC) was prepared by a one-pot reaction method (Davin and Lewis, 2005, Ge and Li, 2018, Liu et al., 2019).Co-monomers including acrylamide and vinyl from treated-grafted polymer were purchased from Sigma-Aldrich as a compatibilizer (Boerjan et al., 2003).All other chemicals were obtained from commercial sources and were of reagent grade.
For the nano-fibrillation of FLC powder, 10 g of untreated LC powder was soaked in distilled water for 48 h.
The suspension of LC powder (1 wt%) was then agitated using a high-speed blender (Vitamix TNC5200, Vita-Mix Corporation, Cleveland, OH, USA) equipped with a 2 L SUS container (X-TREME CAC90B, 143 WARING, East Windsor, NJ, USA) at a stirring speed of 37,000 rpm.After stirring for 45 min, nanofibrillated fiber was obtained by centrifugation at 8,000 × g for 20 min at 25°C.The recovered wet fiber was infiltrated with t-butyl alcohol and freeze-dried (Boerjan et al., 2003, Davin and Lewis, 2005, Ma et al., 2020).
For the pretreatment of FLC powder, 10 g of ligninocellulose (LC) was mixed with 10 g of 3.4dihydroxyacetophenone and 10 mL dimethyl sulfoxide (DMSO as cosolvent) and heated in a dry oven at 110°C for 16 h.The reaction mixture was then suspended in 100 mL of deionized water and centrifuged at 8,000 ×g for 20 min at 25°C.This washing step was conducted five times to remove the 3.4dihydroxyacetophenone/DMSO solution.The product was dried in an oven at 90°C for 24 h and grounded into a powder using a mill.
For alkylation, 5 g of bagasse sample was mixed with 10 g of chloroacetic acid in a 100-mL pressure-proof autoclave reactor (microwave synthesis and extraction, MW-US-UV syn, Co. Ltd., Nagoya, Japan).The reactor was heated in a rotary oven at 140°C.After heating for 3 h, the modified FLC was washed three times with acetone and then twice with distilled water.To remove unreacted chloroacetic acid, FLC acetic acid was centrifuged at 13,000 × g for 20 min at 25°C followed by removal of the supernatant.To prepare the composites, a mixture of 18.8 g of thiosemicarbazide and 1.2 g of FLC acetic acid in 500 mL of xylene was stirred at 130°C for 1 h to obtain a homogeneous solution.The suspension was cast onto a tray covered with polyamide film.After the evaporation of xylene at room temperature in a well-ventilated hood, the resultant dry mixture (thiadiazole biofilm of FLC sample = 47:3:50) was cut into pieces and compounded using a co-rotating twin-screw micro extruder with a recirculating channel (MC5, Xplore Instruments, Sittard, Netherlands) for 3 min at 180°C and 60 rpm.The extruded strands were cooled in air and pelletized.The pellets were injectionmolded into dumbbell-shaped specimens (JIS K7161) (Japanese Standards Association, 2014).The injection and mold temperatures were 190°C and 100°C, respectively (Baraka et al., 2023, Gao and Fatehi, 2019, Ge and Li, 2018, Liu et al., 2019, Ma et al., 2020, Nassar et al., 2023).

Plasma treatment
An alternating current (AC) plasma system was used to coat the seeds.The device generates reactive ion species in gases such as argon, helium, oxygen, and nitrogen at low temperature under a low vacuum (25-90 Pa).Air gas can produce different reactive species like nitrogen, oxygen, carbon and OH groups to enhance the surface of the coated seeds and protect it (Elgendy, 2019, Khalid Buraa et al., 2019).These species react with the seed's surface to generate different chemical entities that can act as a fertilizer and protectant, including making the seeds more resistant to salinity.In addition, using air gas under low vacuum is more chemically effective and The ion flux and ion energy of the reactive species, such as nitrogen, oxygen, and helium, can be detected.The ion flux and energy (Eq. 1 and 2) were calculated using the following equations.
Ion flux: Ion energy: where   is plasma density,   is thermal energy, and M is the ion mass of the reactive species.The ion energy of reactive air species in our study such as nitrogen, and oxygen work at lower energy (2.1 -5 eV) which may be more effective for soft biological treatment and not harmful for our seed treatments.

Wettability measurements
images of all treated and untreated grains in the same frame were taken using a standard stereomicroscope.The diffuse area of the droplets on the surface indicates wettability.
Where,   : is the solid surface tension,   : is the liquid surface tension,   : A solid -land boundary surface tension:

Scanning electron microscopy (SEM)
The morphology of the seeds was studied using a Quattro ThermoFisher SEM equipped with a field emission gun (FEG).SEM images were recorded at an accelerating voltage of 5 kV using a secondary electron detector after sputter coating the seeds.

X-ray photoelectron spectroscopy (XPS)
To determine the elemental composition and the chemical state of the seeds, an XPS system (Kratos Axis Supra equipped with a monochromatic Al Kα X-ray source (hν = 1486.6eV) operating at a power of 75 W and under UHV conditions in the range of ∼10−9 mbar was employed.All spectra were recorded in hybrid mode using electrostatic and magnetic lenses and an aperture slot of 300 μm × 700 μm.The wide and high-resolution spectra were acquired at fixed analyzer pass energies of 80 eV and 20 eV, respectively.The samples were mounted in floating mode to avoid differential charging.

Nuclear Magnetic Resonance (NMR) spectroscopy
All NMR spectra were recorded using a Bruker 400 MHz AVANACIII NMR spectrometer equipped with 4 mm Bruker MAS probe (BrukerBioSpin, Rheinstetten, Germany).The seeds were ground to fine powder using an electrical grinder, then the grain powder was packed in to 4mm zirconium oxide MAS rotor.To create comparable data all experiments were recorded under the same conditions using the same instrumental parameters described previously (Alkordi et al., 2015, Chisca et al., 2014).The data acquisition and analyses were performed using Topspin 3.5pl7 software (Bruker BioSpin, Rheinstetten, Germany).

Germination and seedling vigor test
Two tested cultivars (Misr-1 and Gemmeza-11) were placed on 15 cm diameter petri lined with filter paper (#1 Whatman International, Maidstone, UK) as the media.The filter paper was initially moistened with 7 mL of distilled water, and an additional 5 mL of water was added on the fifth and ninth days of the experiment.Four plasma exposure times were applied plus the control (D(control), D(00), D(22), D(33), and D(44)).For each cultivar, the plasma treatments were arranged in a completely randomized design with four replicates in ambient laboratory conditions (20 ±1 ºC).Full germination was obtained for all tested treatments (100%).After15 days, the seedlings were isolated to measure the radical length, plumule length and seedling length.Seedling dry weight was recorded after oven drying at 105 ºC for 24 h.

Greenhouse trial and physiological studies
At the Faculty of Agriculture, Ain Shams University, the plasma-treated wheat seeds were planted in pots using the randomized complete block design in four replicates.On 2 December 2021, five seeds of wheat cultivars Misr-1 and Gemmeza-11 were placed in plastic pots of 30-cm diameter and filled with 6 kg soil and subjected to the above plasma doses.At 60 days of age, plant leaf samples were taken to estimate chlorophyll a (ch-a), chlorophyll (ch-b), total chlorophyll, carotenoids, malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels following previous reports (Arnon, 1949;Madhava Rao and Sresty, 2000;Rizk et al., 2019;Velikova et al., 2000).

Statistical analysis
Data were subjected to an analysis of variance (ANOVA) test according to Casella (2008), using Costat software, version 6.303 (CoHort Software, Monterey, CA).Based on the randomized complete block design in four replicates, plasma treatment and wheat cultivar were considered as fixed effects, and replications (blocks) were considered as random effects.Mean separation was performed only when the F-test showed significant (P≤0.05)differences among the treatments based on Duncan's multiple range test.

Nonthermal Plasma Composition
To test the properties of the NTP used during the coating process, we performed spectroscopic analysis of the air during coating at different applied voltages.This analysis revealed that, as expected, higher voltage levels generated higher levels of ionization, resulting in higher emission intensity (Figure 1, left panel).Furthermore, we also observed, as expected, that higher voltage led to a higher concentration of reactive species such as nitrogen, oxygen, and iron in our plasma (Figure 1, right panel).Based on these results we conducted all our NTP treatment experiments using 400 V, as these conditions corresponded to suitable reactive species that could interact safely with seeds

Effect of plasma dose on wheat seedling growth rate, germination and vigor
Next, we tested the effect of the NTP dose on the growth status of wheat seedlings.We evaluated four different NTP treatment times (1, 2, 3 and 4 minute exposures), and compared those results to the ones obtained under "no treatment" conditions.As described in Methods and Materials, seeds of each cultivar (Misr-1 and Gemmza-11) were grown under laboratory conditions for 15 days.The seedling growth was measured at regular intervals, and calculated at day 15 (Table 1).We also calculated a correlation coefficient to evaluate the linear relationship between NTP does time and the seedling growth rate (Table 1).We observed that 2 minute NTP treatment produced optimal results in both cultivars.We also noted that 1 minute treatment was not sufficient to reach the maximum beneficial effect (seen at 2 minutes); however, even the 1 minute treatment resulted in some improvement over "no treatment" conditions.Additionally, both 3 and 4 minute treatments led to some deterioration of growth rates, when compared to the 2 minute dose.We also observed some possible differences between the two cultivars, given that Misr-11 exhibited differences between 3 and 4 minute treatments, whereas Gemmeza-11 did not.These results suggest that conclusions based on one cultivar may not translate to others, and that each cultivar needs to be individually evaluated to determine the most optimal treatment conditions.
To further examine the effects of different NTP treatment doses on wheat seeds and their ability to germinate and produce a healthy seedling, we also examined seed germination and seedling vigor.Therefore, five seeds of each wheat cultivar treated with different plasma conditions were planted in petri dishes.After one-month, radical length, plumule length, seedling length and seedling dry weight were measured.Figure 2 show such parameters for Misr-1 and Gemmeza-11 cultivars, respectively.For Misr-1, just the treatment with lignocellulosic biomass (D(00)) was sufficient to result in increases in radical length, plumule length, seedling length and seedling dry weight of 7.9%, 18.1%, 12.0% and 19.7%, respectively (Figure 2A shows just the effects on seedling length and dry weight; results regarding radical length and plumule length are shown in Supplemental Information, as is the Table 1 with exact values from which we derived the % values mentioned in the text).In these experiments with Misr-1, the best increases under NTP dosing conditions were obtained with D(33) (1.2%, 25.0%, 10.9% and 28.1%).For Gemmeza-11 (Figure 2B), we observed more dramatic effects on seedling length, as D(00) increased the values by 51. 4%, 3.9%, 29.3% and 12.0%, respectively, and D(22) increased them to 46.2%, 9.0%, 29.0% and 19.7%.For both cultivars, D(44) was detrimental for their growth, and, additionally, Gemmeza-11 appeared to be especially sensitive to optimal treatment.Taken together, these studies showed that combined treatment of wheat seeds with recycled tree waste (lignocellulosic biomass) and low-pressure plasma (NTP) has the potential to improve seedling growth rates, germination and vigor.

Wettability
To identify potential reasons for the improvements which observed in our seedling studies, the seed surface wettability, a very important parameter for germination, was assessed.Thus, improving and increasing the seed surface wettability is a desirable step in seed treatment approaches.Thus, we conducted seed wettability experiments to examine whether our seed treatment strategy had in impact on this parameter.The contact areas are shown for Misr-1 in Figure 3A and for Gemmeza-11 in Figure 3B (see also Table 1).Misr-1 seeds treated with a 2 minute NTP dose (D( 22)) had reduced wettability compared to D(control) and D(00).In contrast, D(33) or D(44) treated samples had increased levels of surface wettability.On the other hand, Gemmeza-11 seeds had fluctuating wettability levels.The more fluctuating results of the Gemmeza-11 show that its higher sensitivity to plasma treatment when compared to Misr-1, which is aligned with what we observed above in our seedling experiments.
To quantify the seed wettability, we calculated the values of water droplet contact areas on the surface of controlled and plasma treated seeds Misr-1 and Gemmeza-11 (Table 1).In agreement with our microscopy based observations, we noted that the water/seed contact area is significantly decreased in Misr-1 under D( 22) dose conditions, and that effects in Gemmeza-11 are much more variable across the NTP treatment conditions.
From these observations, we concluded that the exposure time affects wettability and nutrient accumulation.

Solid State Nuclear Magnetic Resonance (NMR) characterization of treated seeds.
We wanted to examine whether our combined treatment with recycled tree waste (lignocellulosic biomass) and low-pressure plasma (NTP) led to specific structural and chemical changes on the seed.For overall seed chemical composition evaluation, we chose to apply solid-state Nuclear Magnetic Resonance (NMR) spectroscopy.NMR spectroscopy is a powerful analytical tool that has been extensively used to study chemical composition and molecular identity (Emwas et al., 2020, Kwan andHuang, 2008).The main advantages of NMR spectroscopy are that it is a nondestructive and highly reproducible method where samples can be studied in both solid and liquid states (Chandra et al., 2021a, Chandra et al., 2021b).In the current study we employed solid state NMR spectroscopy to compare the selected treated seeds with the control ones.Given that solid state NMR has not previously been used for wheat seed analysis, we had to develop a sample preparation procedure that would retain seed integrity and allow for accurate acquisition of the NMR data (see sample preparation description in Materials and Methods section).As seen in supplementary materials Figure 1, the seed samples yielded well-disperse solid-state NMR spectra, clearly showing the distribution of the signals corresponding to chemical nature of carbon (aliphatic region between 0 and 100 ppm; aromatic region above 150 ppm) (Li et al., 2016, Khan et al., 2014).We noticed that the treated sample (D(11) in red; supplementary materials Figure 1) displayed a slight increase in NMR peak intensity in aliphatic region where signals from alkyl groups can be found (0-50 ppm) and in the aromatic region (around 175 ppm) when compared to the control (in blue; supplementary materials Figure 1).These small differences in the peak's intensity might be associated with the coating layer of lignin on the surface of the treated wheat seeds, which may improve seed resistance to pathogens or drought, as described before, and is in agreement with improvements we described above.

SEM analysis of Misr-1 and Gemmeza-11 cultivar seeds
To investigate whether the treatment changed the structure of the seed's coat in a manner that may explain improvements in seedling growth rates, germination and vigor, we used scanning electron microscopy (SEM).
This technique allowed us to visualize the finer structural detail of the wheat seed surface, and compare the features of the two cultivars under different treatment conditions.Figure 4 shows SEM images of the Misr-1 samples, including the untreated seed surface at different scales.For D(00), a very thin coating is observed at the top of the seed with 1-10 µm particles (arrows in Figure 4b-b'').For D( 22), a coating of about 30-µm thickness covers almost the whole seed (arrows in Figure 4c-c'').D(33) resulted in a thin coating covering a few hundred micrometers of the seed surface (Figure 4d-d'').For D(44), the coating is barely seen, and only some small particles of a few micrometers in size are observed at the top surface (Figure 4e-e'').Therefore, we conclude that for Misr-1, D( 22) conditions produced the most extensive coating, which may explain the superior seedling growth rate that we noted above.Figure 5 shows SEM images of Gemmeza-11 samples.For D(00) and D(44), a significant coating of about 50 µm was identified at the top surface of the seeds (arrows in b-b'' and e-e'').For D(22) and D(33), the coating was very thin, and only a few particles of about 1 µm in size were present .Therefore, for Gemmeza-11, the link between coating and the results of the seedling growth tests do not seem to be clear at this point.Moreover, these results further highlight the cultivar-dependent response to the coating process.

XPS analysis of Misr-1 and Gemmeza-11 cultivar seeds
Lastly, to gain deeper insights into how the treatment changed the chemical composition of the seeds' surface, we employed X-ray photoelectron spectroscopy (XPS).XPS is a technique that allows elemental analysis of surfaces with high sensitivity.XPS spetra of Gemmeza-11 samples indicated that all seeds have a very similar composition independent of NTP exposure time (Figure 6).However, compared to the control, samples exposed to NTP showed a slight decrease in carbon content and an increase in oxygen content.Also, there was a significant decrease in N content for D00 with respect to other samples.In terms of the elemental content, C, N, In comparison, Misr-1 showed some different trends (Figure 7).Again, the main composition of NTP-exposed samples was more than 97% C, N and O, and 100% for the control seed indicating potential variability in sed surface composition between cultivars.However, there was some variations in the C/O ratio after exposure to NTP.Misr-1D(00) had the highest C/O ratio (and thus the lowest oxygen content), whereas Misr-1D( 22) had the lowest.The spectra also confirmed the insertion of several elements at low concentration to the wheat surface following NTP exposure.In contrast to Gemmeza-11, where the chemical structure was very similar for all samples irrespective of the exposure, the Misr-1 samples showed clear dose-dependency (Figure 7).

Greenhouse trial and physiological studies
Finally, we wanted to examine how different treatments affect the development and physiology of the resulting plants.Based on the above observations, we selected to test seeds from both cultivars (Misr-1 and Gemmeza-11) and expose them to three plasma doses (D00, D22 and D33).We planted the seeds as described in Materials and Methods and grew them under greenhouse conditions as specified.We harvested the leaves after 60 days, and

Discussion
Plasma exposure can change seed-surface properties and stimulate seed germination and seedling growth, induce changes in metabolic plant pathways, modulate enzymatic activities and phytohormones, induce stress resistance, and ultimately influence crop productivity (Han et al., 2016, Holc et al., 2021, Jeevan Kumar et al., 2015, Mildaziene et al., 2021, Scholtz et al., 2021, Šerá et al., 2021, ten Bosch et al., 2017).Plasma treatment affects the wettability of the coated seed.Investigations of plasma-induced changes in plant physiology and biochemistry are likely to reveal new facts of fundamental and applied interest (Mildaziene and Sera, 2022).Our findings indicate remarkable responses of wheat seeds and seedlings to plasma.Since the glow discharge of cold plasma has ions that cause strong surface etching, plasma treatment of wheat seeds (Misr-1 and Gemmeza-11) resulted in chemical re-structuring of the coated surface.In addition, glow plasma contains a higher concentration of relatively aggressive reactive chemical species of higher energy as well as vacuum ultraviolet radiation, which may explain why variations in the nutrient accumulation on the seed coat surface we observed depended on the plasma treatment.
The results indicate also that the response of seeds and seedlings to plasma process cannot be generalized.This is because every seed has its own structure, and so exposure to the same conditions will have different effects.
Additionally, each wheat cultivar has distinct coping mechanisms and interactions with highly reactive oxygen plasma species.Therefore, the different observed responses to plasma could be attributed to the diversified chemical structures of the seed surface and seed size, in addition to the genetic makeup (Zahoranová et al., 2016).The seedling growth rate measurements showed that Gemmeza-11 cultivar is more sensitive than Misr-1 to plasma treatment.In this context, the Gemmeza-11 seedling length and weight were lower after plasma treatment for 3 min based on the reduction in MDA and H2O2 levels.Misr-1 showed better responsiveness and adaptability to cold plasma treatment.Our findings revealed that seed properties can be optimized by optimizing plasma dose to ensure that the coating treatment matches the needs of a specific cultivar.Further studies are needed to examine whether different attributes (i.e.resistance to bacteria or resistance to drought or salinity) require treatment with different plasma doses.
In addition to investigating the effects of the NTP treatment dose on wheat seeds, we also co-treated the seeds with a low molecular-mass biopolymer containing lignin-cellulose.This biopolymer had a major impact on the wetting-drying cycle of the wheat seeds tested here.In previous studies, after 10 wetting-drying cycles, water stability increased 1.5-20 times depending on the soil.The addition of a low molecular-mass biopolymer containing lignin-cellulose exposed under D( 22) after a single wetting-drying cycle increased the water stability more than 200 times.Wettability is not the only factor that influences seed germination.Others include pH, salt concentration, osmotic potential, C, O and N content, bulk density, porosity, and particle size distribution, and these should be considered in lignin-cellulose biofilm design.
Finally, plasma treatment is known to improve the surface hydrophilicity and water uptake of seeds.These effects are mediated by the functionalization and etching of the seed surface (Attia et al., 2019, El-Hashash et al., 2018, El-Hashash and Rizk, 2017, Rizk et al., 2019, Rizk et al., 2018).We observed these effects on the seed surface structure using SEM analysis, which showed that a significant coating of lignin is around 30 µm.For Misr-1, NPT at D(00) and D(22) resulted in resistance to soil salinity.A thinner thickness required a longer plasma dose (D(33)), but too long a dose (D(44) and D(55)) resulted in less coating and less resistance to salinity.For Gemmeza-11, a coating of about 50 µm plasma was effective at D(00) and D(22).

Conclusions
The current work presents the first report of combining the use of organic materials extracted from tree waste residues with plasma treatment for seed coating.This seed coating approach offers many advantages for seeds.
The coating provides a protective layer that helps adaption in harsh conditions such as high salinity.The organic layer also helps to absorb water and preserve it around the seeds, which helps in germination.Also, this layer forms nutrients for the plant.It acts as a fertilizer for the plant and a food for the bacteria that secrete nutrients for the plants.Thus, the treating seed with both lignocellulose and cold plasma may contribute to the production of seeds robust against undesirable environmental conditions, and open opportunities for the mass cultivation of strategic crops like wheat in stressed environments such as saline soils.Further investigations should be adopted to apply the promising findings of plasma plus lignin coated seeds under open field experimentation.
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and O elements account for about 95-98% of the composition of each sample irrespective of NTP exposure time, and for 99% of the untreated sample.NTP led to the inclusion of several elements on the wheat surface, but the overall concentration of these elements remained relatively low.The chemical structure of Gemmeza-11 samples did not undergo significant changes following NTP exposure according to the C 1s high-resolution spectra (See supplementary materials Figure2and 3), whose peak shape was very similar for all samples.The main carbon bonds assigned to C-N, C-O (detected at around 286 eV), C=O and O-C=O (detected at around 288 eV) exhibited similar shapes and intensities independent of the NTP exposure.
The main chemical bonds assigned to C-N, C-O (detected at around 286 eV), C=O and O-C=O (detected at around 288 eV) were less pronounced for samples D(00) and D(44) compared to other doses.This observation is consistent with the lower O and N content of both samples, as shown by the atomic percentages shown in the XPS spectra (Figure 7).
analyzed their chlorophyll a (ch-a), chlorophyll (ch-b), total chlorophyll, carotenoids, malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels.As shown in Table2, D(00)-treated Misr-1 cultivar seeds showed the most ch-b, D(22) showed the most ch-a, and D(33) showed the most H2O2.For Gemmeza-11 cultivar, D(22)      showed the highest content of carotenoids, MDA and H2O2, and D(33) resulted in the highest values for ch-a, ch-b and total chlorophyll.NPT treatment showed positive effects on photosynthetic pigments (ch-a, chlorophyll, total chlorophyll, and carotenoids), as well as enzymatic-protective antioxidants (malondialdehyde and hydrogen peroxide).Future open-field trials should be carried out under various abiotic stresses, such as salinity, to assess the effects of plasma on seed robustness.

Figure 1 .
Figure 1.Spectroscopic analysis of AC plasma of air during coating at different applied voltages.(Left) The panel shows the effect of the applied voltage on plasma intensity and composition.As expected, the intensity and diversity of species generated increased with increased voltage, which is also seen in the panel on the right.

Figure 2 .Figure 3 .
Figure 2. Seedling growth of Misr-1 (A) and Gemmeza-11 (B) wheat cultivars as affected by plasma dose treatments measured after one month of growth in a petri dish Values are the mean of 3 replicates ± standard errors.Points with different letters are statistically significant at p  0.05
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